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Notes: The authors studied the rat VP V1b receptor (V1bR) gene including the 826 base 5´ UTR, which has five ORFs upstream (uORF) of the V1bR protein start codon, to examine the effect of the upstream peptides on V1bR translation. The V1bR gene was cloned into the pALTER®-MAX Vector, and substitution mutations were created in the uORF translation initiation codons with the Altered Sites® II in vitro Mutagenesis System. One microgram of the linearized pALTER®- V1bR construct was transcribed using the Riboprobe® System and translated using Wheat Germ Extract with or without 35S-methionine. The unlabeled protein was analyzed by Western blot. The radiolabeled protein was spun through a 3% sucrose cushion, precipitated and separated by SDS-PAGE. For a possible membrane-targeted protein, the uORF1 was transcribed in vitro and then translated using Rabbit Reticulocyte Lysate, Canine Pancreatic Microsomal Membanes and 35S-methionine. The proteins were spun through a 3% sucrose cushion and then run on a 15% SDS-PAGE gel to determine membrane presence. (3749)

Notes: The Wheat Germ Extract was used to demonstrate that the 3’ end of a viral mRNA acts as a tRNA-like structure (TLS). The tRNA-like structure was shown to accept a [H3]valine residue in the Wheat Germ Extract. The turnip yellow mosaic virus (TYMV) TLS structure was also shown to be able to function with ribosomes by donating [H3]valine in translation assays using Wheat Germ Extract with TYMV RNA but not BMV or AMV RNA. RNAs to be used as templates in the reactions were transcribed with the T7 RiboMAX™ Large Scale RNA Production System. After transcription DNA templates were removed with RQ1 RNase-Free DNase. (3386)

Notes: Messenger RNA was in vitro transcribed and capped using linearized plasmids containing the genes for preplastocyanin and mature plastocyanin, a copper binding protein. Then, 0.2 µg of each plastocyanin RNA was added to Wheat Germ Extract for translation. Translated proteins were labeled with 35S-methionine. (3108)

Notes: In this paper, Promega Wheat Germ Extract System was used to express the oncogene product c-Abl and a mutant (Abl-PP). These in vitro translated proteins were assayed by Western blotting and immunoprecipitation. The Dual-Luciferase® Reporter Assay System was also used during the course of these experiments. (2493)

Notes: The authors investigated expression of BRCA1 from alternative transcripts possessing different 5' UTRs. RT-PCR using Promega's AMV reverse transcriptase was performed to generate cDNAs from total RNA isolated from human tissue. Promega's Taq DNA Polymerase was used for the PCR reaction. The authors also prepared two mRNAs that contained one or the other of the 2 alternative 5' UTRs (ex1a or ex1b) fused with the luciferase coding region. To generate the corresponding DNA for these constructs, the luciferase coding region was amplified from Promega's pGEM vector and ligated to PCR amplified ex1a or exlb. These ligation products served as the template amplifying two cDNA constructs (exla-luc or ex1b-luc).Ten additional cDNAs were made containing a variety of changes to the 5'UTR region of BRCA1. In vitro translation experiments were performed to determine how the composition of the 5'UTR affected protein expression levels (using Promega's RNasin to protect transcribed mRNA; T7 polymerase buffer; and rabbit reticulocyte and wheat germ extracts for translation). The authors conclude that secondary structures associated with elements in the longer 5'UTR reduced translation rates and could be responsible for the reduced expression of BRCA1 often associated with spontaneous ovarian and breast cancers. (2441)

Mol. Cell. Biol.20(12), 4340-4349.
Aca1 and aca2, ATF/CREB activators in saccharomyces cerevisiae, are important for carbon source utilization but not the response to stress2000

Garcia-Gimeno, M.A. and Struhl, K.

Notes: Two yeast DNA binding proteins were translated using Wheat Germ Extract, and assayed for binding to an optimal binding oligo using EMSA. The binding conditions are given in the materials and methods. (2148)

Notes: Control regions of a naturally occurring dicistronic promoter from Cricket Paralysis Virus (CrPV) were cloned into a previously described dicistronic reporter vector coding for both Fluc and Rluc. The reporter was used either for in vitro translation from RiboMAX™-produced uncapped RNA (RiboMAX™ Large-Scale RNA Production System) using either an RRL or WGE system, and measuring the translation using the Dual-Luciferase® Reporter Assay System, or for transfection into SL-2 cells. RNA was also used for transfection of the SL2 cells, and luciferase activities were measured using DLR. Luciferase readings are reported as ratio of Fluc activity to Rluc activity, with the no insert vector ratio set to 1. (2147)

Notes: Several proteins were translated using the TNT® Coupled Wheat Germ Extract System. p105 was expressed, then used as a substrate for ubiquitination, with the E1 protein necessary for the process contributed by the WGE. (2157)

Notes: Recombinant HNF-6 was expressed using the TNT® Coupled Wheat Germ Extract System. The resulting protein was used in a gel shift assay, using 5µl of the reaction in a total volume of 20µl. (2156)

Notes: BRCA2 was screened for mutations in the ovarian cancer cluster region by means of the protein-truncation test (PTT). PTT was performed with the TNT® Coupled Reticulocyte Lysate System, incorporating [35S] methionine for protein detection. (0343)

EMBO J.16, 659-671.
A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state.1997

Lee, G. J. , Roseman, A. M. , Saibil, H. R. , Vierling, E.

Notes: The Quantilum™ Recombinant Luciferase was heat denatured in the presence of heat shock protein hsp 18.1 and the renaturation of the protein was assayed in with the Luciferase Assay System. The denaturation was performed in either Rabbit Reticulocyte Lysate or Wheat Germ Extract in the presence or absence of added ATP. (0809)

Notes: Dual-Luciferase® Reporter Assay System studies were performed in HepG2 cells. The experimental plasmid constructed in pGL2-Basic Vector was used at a 40:1 ratio over the pRL-CMV Vector. The experimental and control plasmids were cotransfected with a third plasmid expressing hepatocyte nuclear factor 6 from a CMV promoter. The luciferase activity expressed in the presence or absence of the hepatocyte factor were measured. The TNT® Coupled Wheat Germ Extract System was used to in vitro translate four transcription factors for assay of gel shift with the promoter construct. (0870)

Notes: This paper describes a method for quantitation of luciferase mRNA by in vitro translation using Promega TNT® Coupled Wheat Germ Extract System. (Rabbit reticulocyte lysate could not be used because of luciferase quenching problems). In this reporter gene assay, COS cells were transfected with a firefly luciferase reporter plasmid driven by promoters/enhancers of varying strengths and total cellular RNA was isolated and translated in vitro using the TNT® System. To normalize expression, a CMV Renilla luciferase or beta-galactosidase vector was used as a control. To measure luciferase activity either Promega Luciferase Assay System or Dual-Luciferase® Reporter Assay System was used. (2047)

Notes: Expression of luciferase was studied in the TNT® Coupled Wheat Germ Extract System in the presence of circular and linear antisense (sulfide cross-linked) oligonucleotides that contained sequences complementary to the initiation codon region of firefly luciferase mRNA. Satisfactory antisense properties were displayed with modified circular oligonucleotides that contained a short random mismatching sequence. The application of these molecules to inhibition or preservation of enzymatic reactions may be useful for designing antigene technologies. (1486)

Notes: The catalytic subunit from the Na+-K+-ATPase was expressed in Wheat Germ Extracts or Rabbit Reticulocyte Lysates. In immunoblots using an antibody that is specific to the first 9 residues of the predicted protein, the in vitro synthesized protein but not the in vivo synthesized protein was detected, suggesting that the protein is processed in vivo. Processing was inefficient or absent in the TNT® Coupled Reticulocyte Lysate Systems. (0546)

Biochemistry35, 3614-3618.
Cryptic initiation at the human D4 receptor reveals a functional role for the amino terminus.1996

Schoots, O., Sanyal, S., Guan, H.C., Jovanovic, V., Van Tol, H.H.

Notes: The TNT® Coupled Reticulocyte Lysate System used an alternative initiation codon when translating 5´ deletion mutants of the human D4 receptor. This result suggests that an amino-terminal truncated D4 receptor might exist in vivo and that the amino terminus may stabilize the active state of the receptor. (0415)

Notes: Luciferase mRNA was transcribed and the mRNA was translated in a Wheat Germ Extract System. Luminescence was measured by adding luciferin to the translation mixtures before incubation. The accumulation of full-sized luciferase correlated with the increase in light production, and translation arrest lead to immediate cessation of luciferase activity increase. The ribosome-bound protein had no detectable enzymatic activity, but enzymatic activity was shown a few seconds after luciferase was released from the ribosome. Renaturation of denatured luciferase under these conditions had a half-time of approx. 14 minutes, supporting the cotranslational folding hypothesis (peptides start to attain native structure while synthesizing on the ribosome). (0724)

Mol. Cell. Biol.16, 1169-1178.
Interaction of the v-Rel oncoprotein with NF-kappaB proteins: Heterodimers of a transformation-defective v-Rel mutant and NF-kappaB p52 are functional in vitro and in vivo.1996

Notes: Native and mutated AR cDNAs were transcribed with the TNT® T7 Coupled Reticulocyte Lysate System. The reaction mixtures were divided and supplemented with either androgen or vehicle. For EMSAs, the preincubated receptors were placed in binding reactions with [32P]-labeled dsDNA corresponding to a glucocorticoid/androgen-responsive element (ARE). All AR receptors with intact DNA-binding domains formed specific complexes with the ARE. The presence of active hormone altered the mobility of receptor-DNA complexes probably due to a conformational change. Mutant proteins could form heterodimers with native AR that interacted with DNA. Androgen receptor (AR) was translated using the Rabbit Reticulocyte Lysate System, and RelA was translated using Wheat Germ Extract. Coimmunoprecipitation was performed with anti-AR or anti-RelA, but no specific association between AR and RelA was detected (even with the cross-linking agent dithiobis(succinimidylpropionate) present). This finding does not exclude the possibility that AR-RelA interactions occur in vivo. (1669)

Notes: The Protein Truncation Test (PTT) is investigated as a rapid, first mutation screening approach in exon 11 of the BRCA1 gene. Eighty-one percent of the mutations found in 63 patients are truncation mutations, and 49% of these mutations are in exon 11 so the PTT is a sound screening method. The Wheat Germ Extract performed better than the Rabbit Reticulocyte Lysates with the primer set used. The investigators suggest that the choice of translation be empirically determined for each primer set used. (0538)

Notes: Retinoid X-receptor was transcribed and translated in vitro using the TNT® Coupled Wheat Germ Extract System. The protein was used for EMSAs with Ap-1 and VDR responsive regions from the human osteocalcin gene. Both half sites of the VDRE are required for VDR-RXRalpha binding, suggesting that RXRalpha is not the physiological accessory factor for VDR-VDRE interactions. The RXRalpha protein was also produced in the presence of [35S]methionine and analyzed by SDS-PAGE. (1805)

Notes: c-Jun, c-Jun-bZIP and cJun-TAD were synthesized using the TNT® SP6 Coupled Wheat Germ Extract System. The proteins were used in GST binding assays with GSTGal4, GSTGal4(1-147) and GSTGal4/CR1. These assays show that Gal4 and c-Jun can physically interact in vitro. Gal4(1-147) increases the binding of c-Jun to a consensus AP1 DNA binding site in EMSAs. (1813)

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